The use of site-specific DNA recombinases has expanded the spectrum of genetic manipulations that can be carried out in both prokaryotic and eukaryotic organisms. While various site-specific DNA recombinases, such as the yeast-derived Flp/frt, are becoming increasingly popular, the Cre/loxP system is currently the most widely used system. Because of its simplicity and versatility, Cre has found widespread use in conditional mutagenesis and gene expression, gene replacement and deletion, and chromosomal engineering experiments.
Cre is a site-specific DNA recombinase derived from the P1 bacteriophage and is a member of the lambda integrase or tyrosine family of site-specific recombinases (1). Members of this family catalyze DNA recombination by a common catalytic mechanism and recognize target recombination sites with similar structural features. In the case of the Cre protein, it recognizes 34 base pair sequences known as loxP sites. The loxP sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats. Cre recombines the 34 base pair loxP DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine.
The P1 genome, the sequencing of which has recently been completed in its entirety (12), is relatively large for a temperate DNA bacteriophage: 95 kb. P1 is unusual among temperate bacteriophages in that it maintains itself as an extrachromosomal unit copy plasmid in the lysogenic state. Cre is expressed in P1 lysogens and its site-specific DNA recombination activity contributes to the stable maintenance of the P1 prophage during lysogeny. Cre resolves P1 dimers that arise by homologous recombination after DNA replication, thus helping to ensure segregation of a P1 monomer to each daughter cell at cell division (13).
The P1 Cre gene and its 34 bp recombination target site loxP lie in a relatively short interval of P1 DNA that includes two other phage functions with unusual features. To the left of Cre is the immC immunity region of P1 that encodes the C1 repressor and several other immunity proteins that modify its action. ImmC, in turn, lies just to the right of the two genes for the P1 pacase or terminase and the pac site at which P1 DNA packaging begins. DNA packaging in P1 is unusual because protein recognition of the P1 DNA packaging site is regulated by DNA adenine methylation (dam). Although immunity in P1 is orchestrated in a complex manner and includes several different immunity regions (16), including antirepressor components, the C1 protein is unusual compared to other phage repressors because it recognizes an asymmetric DNA binding site (17). To the left of the P1 Cre gene is c8, another immunity gene, followed by ref, a gene involved in the homologous recombination of short DNA repeats (18,19).
Several P1-related phages also maintain themselves as an extrachromosomal plasmid in the lysogenic state, but comparable recombinase function in these P-1 related phages has yet to be elucidated. In particular, Cre homologues that perform site specific recombination at sites distinguishable from the lox site have not been previously characterized.